Jose R. Conejo-Garcia

Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival

Regulatory T (Treg) cells mediate homeostatic peripheral tolerance by suppressing autoreactive T cells. Failure of host antitumor immunity may be caused by exaggerated suppression of tumor-associated antigen–reactive lymphocytes mediated by Treg cells; however, definitive evidence that Treg cells have an immunopathological role in human cancer is lacking. Here we show, in detailed studies of CD4+CD25+FOXP3+ Treg cells in 104 individuals affected with ovarian carcinoma, that human tumor Treg cells suppress tumor-specific T cell immunity and contribute to growth of human tumors in vivo. We also show that tumor Treg cells are associated with a high death hazard and reduced survival. Human Treg cells preferentially move to and accumulate in tumors and ascites, but rarely enter draining lymph nodes in later cancer stages. Tumor cells and microenvironmental macrophages produce the chemokine CCL22, which mediates trafficking of Treg cells to the tumor. This specific recruitment of Treg cells represents a mechanism by which tumors may foster immune privilege. Thus, blocking Treg cell migration or function may help to defeat human cancer.

Tumor-infiltrating dendritic cell precursors recruited by a beta-defensin contribute to vasculogenesis under the influence of Vegf-A.

The involvement of immune mechanisms in tumor angiogenesis is unclear. Here we describe a new mechanism of tumor vasculogenesis mediated by dendritic cell (DC) precursors through the cooperation of β-defensins and vascular endothelial growth factor-A (Vegf-A). Expression of mouse β-defensin-29 recruited DC precursors to tumors and enhanced tumor vascularization and growth in the presence of increased Vegf-A expression. A new leukocyte population expressing DC and endothelial markers was uncovered in mouse and human ovarian carcinomas coexpressing Vegf-A and β-defensins. Tumor-infiltrating DCs migrated to tumor vessels and independently assembled neovasculature in vivo. Bone marrow–derived DCs underwent endothelial-like differentiation ex vivo, migrated to blood vessels and promoted the growth of tumors expressing high levels of Vegf-A. We show that β-defensins and Vegf-A cooperate to promote tumor vasculogenesis by carrying out distinct tasks: β-defensins chemoattract DC precursors through CCR6, whereas Vegf-A primarily induces their endothelial-like specialization and migration to vessels, which is mediated by Vegf receptor-2.

Synthetic lethality by targeting EZH2 methyltransferase activity in ARID1A -mutated cancers

ARID1A, a chromatin remodeler, shows one of the highest mutation rates across many cancer types. Notably, ARID1A is mutated in over 50% of ovarian clear cell carcinomas, which currently has no effective therapy. To date, clinically applicable targeted cancer therapy based on ARID1A mutational status has not been described. Here we show that inhibition of the EZH2 methyltransferase acts in a synthetic lethal manner in ARID1A mutated ovarian cancer cells. ARID1A mutational status correlates with response to the EZH2 inhibitor. We identified PIK3IP1 as a direct ARID1A/EZH2 target, which is upregulated by EZH2 inhibition and contributes to the observed synthetic lethality by inhibiting PI3K/AKT signaling. Significantly, EZH2 inhibition causes regression of ARID1A mutated ovarian tumors in vivo. Together, these data demonstrate for the first time a synthetic lethality between ARID1A mutation and EZH2 inhibition. They indicate that pharmacological inhibition of EZH2 represents a novel treatment strategy for ARID1A mutated cancers.The gene encoding ARID1A, a chromatin remodeler, shows one of the highest mutation rates across many cancer types. Notably, ARID1A is mutated in over 50% of ovarian clear cell carcinomas, which currently have no effective therapy. To date, clinically applicable targeted cancer therapy based on ARID1A mutational status has not been described. Here we show that inhibition of the EZH2 methyltransferase acts in a synthetic lethal manner in ARID1A-mutated ovarian cancer cells and that ARID1A mutational status correlated with response to the EZH2 inhibitor. We identified PIK3IP1 as a direct target of ARID1A and EZH2 that is upregulated by EZH2 inhibition and contributed to the observed synthetic lethality by inhibiting PI3K–AKT signaling. Importantly, EZH2 inhibition caused regression of ARID1A-mutated ovarian tumors in vivo. To our knowledge, this is the first data set to demonstrate a synthetic lethality between ARID1A mutation and EZH2 inhibition. Our data indicate that pharmacological inhibition of EZH2 represents a novel treatment strategy for cancers involving ARID1A mutations.

Polyethylenimine-based siRNA nanocomplexes reprogram tumor-associated dendritic cells via TLR5 to elicit therapeutic antitumor immunity

The success of clinically relevant immunotherapies requires reversing tumor-induced immunosuppression. Here we demonstrated that linear polyethylenimine-based (PEI-based) nanoparticles encapsulating siRNA were preferentially and avidly engulfed by regulatory DCs expressing CD11c and programmed cell death 1-ligand 1 (PD-L1) at ovarian cancer locations in mice. PEI-siRNA uptake transformed these DCs from immunosuppressive cells to efficient antigen-presenting cells that activated tumor-reactive lymphocytes and exerted direct tumoricidal activity, both in vivo and in situ. PEI triggered robust and selective TLR5 activation in vitro and elicited the production of hallmark TLR5-inducible cytokines in WT mice, but not in Tlr5-/- littermates. Thus, PEI is a TLR5 agonist that, to our knowledge, was not previously recognized. In addition, PEI-complexed nontargeting siRNA oligonucleotides stimulated TLR3 and TLR7. The nonspecific activation of multiple TLRs (specifically, TLR5 and TLR7) reversed the tolerogenic phenotype of human and mouse ovarian tumor-associated DCs. In ovarian carcinoma-bearing mice, this induced T cell-mediated tumor regression and prolonged survival in a manner dependent upon myeloid differentiation primary response gene 88 (MyD88; i.e., independent of TLR3). Furthermore, gene-specific siRNA-PEI nanocomplexes that silenced immunosuppressive molecules on mouse tumor-associated DCs elicited discernibly superior antitumor immunity and enhanced therapeutic effects compared with nontargeting siRNA-PEI nanocomplexes. Our results demonstrate that the intrinsic TLR5 and TLR7 stimulation of siRNA-PEI nanoparticles synergizes with the gene-specific silencing activity of siRNA to transform tumor-infiltrating regulatory DCs into DCs capable of promoting therapeutic antitumor immunity.

Ovarian cancer progression is controlled by phenotypic changes in dendritic cells

Dendritic cells are transformed to become immunosuppressive during ovarian cancer progression.

Tumor-associated neutrophils stimulate T cell responses in early-stage human lung cancer

Infiltrating inflammatory cells are highly prevalent within the tumor microenvironment and mediate many processes associated with tumor progression; however, the contribution of specific populations remains unclear. For example, the nature and function of tumor-associated neutrophils (TANs) in the cancer microenvironment is largely unknown. The goal of this study was to provide a phenotypic and functional characterization of TANs in surgically resected lung cancer patients. We found that TANs constituted 5%-25% of cells isolated from the digested human lung tumors. Compared with blood neutrophils, TANs displayed an activated phenotype (CD62L(lo)CD54(hi)) with a distinct repertoire of chemokine receptors that included CCR5, CCR7, CXCR3, and CXCR4. TANs produced substantial quantities of the proinflammatory factors MCP-1, IL-8, MIP-1α, and IL-6, as well as the antiinflammatory IL-1R antagonist. Functionally, both TANs and neutrophils isolated from distant nonmalignant lung tissue were able to stimulate T cell proliferation and IFN-γ release. Cross-talk between TANs and activated T cells led to substantial upregulation of CD54, CD86, OX40L, and 4-1BBL costimulatory molecules on the neutrophil surface, which bolstered T cell proliferation in a positive-feedback loop. Together our results demonstrate that in the earliest stages of lung cancer, TANs are not immunosuppressive, but rather stimulate T cell responses.

ER Stress Sensor XBP1 Controls Anti-tumor Immunity by Disrupting Dendritic Cell Homeostasis

Dendritic cells (DCs) are required to initiate and sustain T cell-dependent anti-cancer immunity. However, tumors often evade immune control by crippling normal DC function. The endoplasmic reticulum (ER) stress response factor XBP1 promotes intrinsic tumor growth directly, but whether it also regulates the host anti-tumor immune response is not known. Here we show that constitutive activation of XBP1 in tumor-associated DCs (tDCs) drives ovarian cancer (OvCa) progression by blunting anti-tumor immunity. XBP1 activation, fueled by lipid peroxidation byproducts, induced a triglyceride biosynthetic program in tDCs leading to abnormal lipid accumulation and subsequent inhibition of tDC capacity to support anti-tumor T cells. Accordingly, DC-specific XBP1 deletion or selective nanoparticle-mediated XBP1 silencing in tDCs restored their immunostimulatory activity in situ and extended survival by evoking protective type 1 anti-tumor responses. Targeting the ER stress response should concomitantly inhibit tumor growth and enhance anti-cancer immunity, thus offering a unique approach to cancer immunotherapy.

Microbially Driven TLR5-Dependent Signaling Governs Distal Malignant Progression through Tumor-Promoting Inflammation

The dominant TLR5(R392X) polymorphism abrogates flagellin responses in >7% of humans. We report that TLR5-dependent commensal bacteria drive malignant progression at extramucosal locations by increasing systemic IL-6, which drives mobilization of myeloid-derived suppressor cells (MDSCs). Mechanistically, expanded granulocytic MDSCs cause γδ lymphocytes in TLR5-responsive tumors to secrete galectin-1, dampening antitumor immunity and accelerating malignant progression. In contrast, IL-17 is consistently upregulated in TLR5-unresponsive tumor-bearing mice but only accelerates malignant progression in IL-6-unresponsive tumors. Importantly, depletion of commensal bacteria abrogates TLR5-dependent differences in tumor growth. Contrasting differences in inflammatory cytokines and malignant evolution are recapitulated in TLR5-responsive/unresponsive ovarian and breast cancer patients. Therefore, inflammation, antitumor immunity, and the clinical outcome of cancer patients are influenced by a common TLR5 polymorphism.

The role of dendritic cell precursors in tumour vasculogenesis.

In this review, we discuss the recent identification in vivo of a population of CD11c+ cells exhibiting simultaneous expression of both endothelial and dendritic cell markers, termed vascular leukocytes (VLCs). VLCs are highly represented in human ovarian carcinomas and, depending on the milieu, can assemble into functional blood vessels or act as antigen-presenting cells. The identification of dendritic cell precursors as bipotent cells has important implications for the physiopathology and therapy of tumours. VLCs emerge as a novel therapeutic target against tumour vascularisation.

Reprogramming Tumor-Associated Dendritic Cells In Vivo Using miRNA Mimetics Triggers Protective Immunity against Ovarian Cancer

Modulating the activity of miRNAs provides opportunities for novel cancer interventions. However, low bioavailability and poor cellular uptake are major challenges for delivering miRNA mimetics specifically to tumor cells. Here, we took advantage of the spontaneous enhanced endocytic activity of ovarian cancer-associated dendritic cells (DC) to selectively supplement the immunostimulatory miRNA miR-155. In vivo processing of nanoparticles carrying oligonucleotide duplexes mimicking the bulged structure of endogenous pre-miRNA (but not siRNA-like oligonucleotides) dramatically augmented miR-155 activity without saturating the RNA-induced silencing complex. Endogenous processing of synthetic miR-155 favored Ago2 and, to a lesser extent, Ago4 loading, resulting in genome-wide transcriptional changes that included silencing of multiple immunosuppressive mediators. Correspondingly, tumor-infiltrating DCs were transformed from immunosuppressive to highly immunostimulatory cells capable of triggering potent antitumor responses that abrogated the progression of established ovarian cancers. Our results show both the feasibility and therapeutic potential of supplementing/replenishing miRNAs in vivo using nonviral approaches to boost protective immunity against lethal tumors. Thus, we provide a platform, an optimized design, and a mechanistic rationale for the clinical testing of nonviral miRNA mimetics.